The present invention relates to a recombinant herpesvirus of turkeys (hereinafter referred to as BVT) and a recombinant Marek""s disease virus (hereinafter referred to as MDV), in which an foreign gene has been integrated into a non-essential region of the genome of HVT or MDV, and vaccines employing said recombinant.
Conventionally known virus vector vaccines that are prepared using gene recombinant technology include vaccines that employ a virus of the genus poxvirus as a vector (Ogawa R. et al., Vaccine, 8:486-490 (1990)), vaccines that employ adenovirus as a vector (HSU, K. H. et al., Vaccine, 12;607-612 (1994)), vaccines that employ baculovirus as a vector, as well as vaccines that employ a virus of the genus herpesvirus as a vector (Shin, M. -F. et al., Proc. Natl. Acad. Sci. U.S.A., 81:5867-5870 (1984)). Among them, recombinant vector vaccines based on the genus herpesvirus are under intensive study in recent years.
As virus vectors that permit the expression of a gene for a foreign antigen, there are known human herpesvirus (HSV), Aujeszky""s disease virus (pseudorabies virus; PRV) (Van Zijl M. et al., J. Virol., 65:2761-2765 (1991)), herpesvirus of turkey (HVT) (Morgan R. W. et al., Avian Dis. 36:858-870 (1992)), Marek""s disease virus (MDV), and the like. Since, HVT virus and vaccine strain MDV, among them, have high safety in poultry that are the subject of vaccination and have good vaccine characteristics, they are attracting attention as vector viruses for avians.
Unlike poxvirus that has a mode of infection in which the virus is once released into the blood from the infected cell and then it infects another call during its infection from the infected cell to another cell, HVT and MDV establish infection via a cell-cell interaction to an adjacent cell. Thus, they are relatively free from the influences of HVT- or MDV-specific antibodies present in the circulating blood.
Conventionally, problems have been recognized in that the efficacy of live virus vaccines is attenuated by the presence of maternal antibody from the mother bird, resulting in the failure of exhibiting their full effects.
In recent years, methods of inoculating vaccines into developing chicken eggs have been developed as one of the methods of vaccination to chickens, and the usefulness of HVT or MDV as a vaccine is gaining recognition.
However, conventionally known genetic regions so far reported for construction of recombinant BVT or MDV for foreign antigen were only genetic regions that are considered non-essential for survival of HVT such as the TK region (Ross L. et al., 16th International Herpes virus Workshop (1991)), the US10region (Sakaguchi M. et al., Vaccine, 12:953-957 (1994)), and the US2 region (Sondermeijer, P. J. et al., Vaccine, 11:349-358 (1993)). Such integration into non-essential regions can attenuate the antigenicity of HVT or MDV since it causes the expression of a foreign gene in stead of a gene that, though non-essential, should naturally be expressed in HVT i.e. a gene that will make an antigenic determinant. In addition, the possibility cannot be ruled out that the genetic machineries (enhancers, promoters, terminators, etc.) involved in the transcription or translation of the open reading frame for the inserted region may adversely affect the expression of the inserted gene.
In fact, there are reports that the deletion of genetic regions encoding proteins that are considered to be non-essential or the integration therein of foreign genes resulted in the modification of viral morphology or the reduction in antigenicity. Furthermore, the integration of foreign genes has been used, in some cases, as a method of preparing attenuated vaccines.
Another report demonstrates that the insertion of a foreign gene into the TK region and the expression thereof resulted in the decreased antigenicity of the expressed gene (Ross L. et al., J. Gen. Virol., 74:371-377 (1993)). This method also has a number of problems as a vaccine in that many antigen genes cannot be inserted since the length of antigen genes that can be inserted into specific ORFs is limited.
As a result of intensive study to solve the above problems, the inventors of the present invention have found that there are the gene insertion regions of HVT or MDV into which a variety of genes for foreign antigen can be inserted and the antigen protein can be stably expressed, i.e. the untranslated region of HVT or MDV mentioned above, that genes of various foreign antigens can be inserted therein, and that by preparing recombinant HVT or MDV into which these genes of foreign antigen have been inserted and then infecting these recombinant viruses to the hosts, adequate vaccination effect cat be conferred to the host, and thereby have completed the present invention.
As a result of intensive study to solve the above problems, the inventors of the present invention have prepared a recombinant virus in which a foreign gene has been inserted into a specific site in a untranslated region of a virus DNA belonging to the avian infectious herpesvirus and found that said recombinant virus can be used as a vaccine, and thereby have completed the present invention.
Thus, the present invention relates to avian infectious recombinant herpesviruses in which a foreign gene has been inserted into a genetic region that is an untranslated region in the genome. Preferably, the virus mentioned above is herpesvirus of turkeys (HVT) and Marek""s disease virus (MDV).
The above untranslated region is preferably an untranslated region present in the open reading frame of herpesvirus of turkeys or the open reading frame of Marek""s disease virus, each corresponding to the open reading frame of human herpes simplex virus. A specifically preferred insertion site for a foreign gene is at least one insertion site selected from the group consisting of sites in (1) between UL44 and UL45, (2) between UL45 and UL46, (3) between UL41 and UL42, (4) between UL40 and UL41, (5) a region located downstream of the gB gene, (6) between UL53 and UL54, and (7) between UL36 and UL37.
The above foreign gene is preferably a gene derived from a pathogen of avian infectious diseases, and most preferably an antigen gene derived from a pathogen selected from the group consisting of viruses, bacteria, fungi, and protozoa. Furthermore, the above foreign gene is preferably a gene derived from a pathogen selected from the group consisting of Newcastle disease virus (NDV), Gumboro disease virus (infectious bursal disease virus: IBDV), infectious laryngotracheitis virus (ILTV), infectious bronchitis virus (IBV), mycoplasma (MG), and coccidia.
The present invention also relates to a chicken vaccine comprising the above recombinant virus as an active ingredient.
The present invention will now be explained hereinbelow in more details.
Viruses for Use in the Present Invention
Of the viruses that infect avians, viruses for use in the present invention are preferably those that belong to the genus herpesvirus (avian infectious herpesvirus), because viruses that belong to the genus herpesvirus have a property of permanently surviving in the body of an infected animal in the state of latent infection or persistent infection.
Of the avian infectious herpesviruses, specifically herpesvirus of turkey or Marek""s disease virus (MDV) is preferred. Their effectiveness as a vaccine can be expected because the above viruses have a long infection period and are expected to confer to avians vaccination effects for a long period of time.
HVT or MDV
HVT or MDV for use in the present invention may include, but not limited to, naturally occurring ones or those available from ATCC etc. with or without charge.
Preferred examples of HVT include those that belong to the gamma herpesvirus subfamily, that are originally nonpathogenic, and that are noncarcinogenic and are used as a vaccine for poultry. Specifically, there can be mentioned FC126 (ATCC VR-584B), PB-THV1, H-2, YT-7, WTHV-1, EPRS-26, and the like. For example, the FC126 strain may be preferably used. Furthermore, specific examples of MDV include CV1988, SR1, and the like.
In order to construct a recombinant virus of the present invention, initially, the above virus is propagated in a suitable host cell and then the genomic DNA is obtained. Then the untranslated region of the genomic DNA is identified and then a foreign gene described below is inserted into the region.
The host and the conditions for propagating the virus are selected as appropriate depending on the virus to be propagated. For example, when HVT is to be propagated, CEF, a embryonated egg, a chicken kidney dell, and the like are used as the host cell. It may be cultured in a culture medium such as Eagle""s MEM, Leibowitz-L-15/McCoy 5A (1:1 mixture) culture medium at about 37xc2x0 C. for 3 to 4 days.
DNA is extracted from the virus infected cells cultured as above according to a conventional method. Thus, the cells grown in monolayers are scraped, and then spun to harvest the supernatant. After protein is denatured in the lysis buffer and removed, DNA is extracted with phenol and ethanol.
The untranslated region of the viral DNA thus obtained may be identified as described below.
Untranslated regions as used herein refer to a region of nucleotides that have no ORF and do not define an amino acid sequence of protein to be expressed by translation, and a region of nucleotides in which the ORF is not involved in any of transcription, translation, or protein expression. The nucleotide sequences contained in this region may be those that have substitutions, deletions, or additions of bases, unless they include an ORF.
Regions for Insertion of Foreign Gene
For the purposes of this invention, regions into which foreign genes are inserted are termed as insertion regions for foreign gene, and sites into which foreign genes are inserted are termed as sites for insertion of foreign gene.
Regions for insertion of foreign gene can be obtained as described below. By way of example, HVT or MDV will be explained.
The untranslated regions of these viruses may be obtained as described below. First, sequences flanking a sequence estimated to be an untranslated region are selected from regions that have been identified, such as human herpes simplex virus type I (HSV-I) of which entire nucleotide sequence has been determined and HVT and MDV-1 sequences that have a high homology therebetween.
Then, DNA primers are synthesized based on these sequences, and a polymerase chain reaction (PCR) is carried out under predetermined conditions using the DNA of HVT or MDV as a template to amplify a specific gene.
The absence of an ORF in the amplified gene is confirmed by DNA sequence analysis and the positions where the genetic machineries (enhancers, promoters, terminators, etc.) involved in the transcription or translation of the ORE are not present are also located, and then insertion regions for foreign gene are determined.
In order to clarify that the region is non-essential for viral propagation and that a foreign gene can be introduced into this region, the addition, deletion, or replacement of a specific sequence is carried out in the region, which is then infected to CEF (chick embryo fibroblast), and changes in infectivity and a propagating property beforehand after such modifications are investigated.
The above-mentioned addition, deletion, or replacement of a specific sequence can be carried out using standard tools such as in vitro mutagenesis, PCR, site-directed mutagenesis, and site-specified mutation as described in Japanese Examined Patent Publication (Kokoku) No. 6-16709.
CEF is infected at m.o.i≈1, grown at 37xc2x0 C. for 3 to 4 days, and then viral propagation, cell morphology, plaque morphology, and cell immortality are observed.
As a result, if it is confirmed that there are no differences in cell and plaque morphology from the strain before nucleotide modification, and that the difference in viral propagation from the strain before nucleotide modification is within xc2x120% as a mean of five repeat runs, it is thereby concluded that said site is a region into which a foreign gene can be inserted (a possible insertion region for foreign gene). The size of the insertion region for foreign gene including the untranslated region and regions flanking it is sufficiently 10 bp or more, preferably 100 bp or more, and more preferably 500 bp or more.
Insertion sites for foreign gene are not limited as long as they are within the untranslated region. Specific examples include sites in (1) between UL44 and UL45 and between UL45 and UL46, (2) between UL41 and UL42, (3) between UL40 and UL41, (4) a region located downstream of the gB gene, (5) between UL53 and UL54, (6) between UL36 and UL37, and the like. They are described for HSV-1 in the Proceedings of the 16th Herpes Virus Workshop (held at Pacific Grove in California, U.S.A., on Jul. 7-12, 1991).
Among them, preferred sites include (1) and (4) for which homology has been confirmed in the ORF in not only HSV-1 but also MDV, and more preferably (1) for which an untranslated region in between ORFs can be estimated.
Construction of Plasmids Containing a Foreign Gene
In order to insert a foreign gene in an untranslated region of HVT or MDV, it is necessary to clone a sequence containing the untranslated region into a plasmid in advance, but the plasmid is not limited.
For example, there can be mentioned plasmids such as pBR322, pBR325, pBR327, pBR328, pUC18, pUC19, pUC7, pUC8, and pUC9, and phages such as lambda phage and M13 phage, and cosmids such as pHC79.
An untranslated region obtained as mentioned above is integrated into these plasmids according to a conventional method.
In order to insert a foreign gene into the untranslated region integrated as above, mutation is carried out at a specific site of the untranslated region that was cloned in a plasmid as above to make a new cleavage site for restriction enzymes, and then the foreign gene is inserted into the site.
A method of carrying out mutation may be a conventional method, and a method commonly used by a person skilled in the art such as in vitro mutagenesis and PCR can be used. Thus, in the PCR method, a mutation such as the deletion, replacement, or addition of 1 to 2 nucleotides in the PCR primer is carried out, and the primer is then used to create a mutation.
For the purposes of this invention, the foreign gene to be inserted herein preferably contains both of a self-derived gene that originally is not included in the region and a non-self-derived gene, and is preferably an antigen gene of an avian infectious herpesvirus.
Examples of such genes include genes derived from a pathogen of avian infectious diseases. Examples of pathogens that cause infection in avians include virus, bacteria, fungi, protozoa, and the like. Antigen genes contained in these pathogens i.e. genes encoding determinants of antigen can be preferably used.
Specific examples of such pathogens include New Castle disease virus (NDV) and Gumboro disease (infectious bursal disease virus) (IBDV) that are lifelong threats to chickens, infectious laryngotracheitis virus (ILTV), infectious bronchitis virus (IBV), mycoplasma (MG), and coccidia that are threats to young and middle age of chickens and after.
Specifically, in diseases in which the genes of neutralizing antigens or the genes of antigens that are considered immunoprotective antigens have been identified, it is possible to integrate these genes into avian infectious herpesviruses such as HVT and MDV and then to express them as antigens in the body of chickens that are infected with the integrated virus. This also makes it possible to use them as effective vaccines.
Specifically, proteins that are expressed in the birds infected with a recombinant virus constructed by integrating a gene into an avian infectious herpesvirus such as HVT or MDV may be any one of the structural protein or the non-structural protein, and are not limited as long as their DNA sequence is known.
For NDV, for example, there can be mentioned HN protein, F protein, and NP protein, and for IBV there can be mentioned M protein, N protein, and spike protein. For IBDV, there can be mentioned the entire protein comprising from VP1 to VP5, for ILTV, since it is a herpesvirus, proteins that are homologous to HSV-1, MDV, or HVT (specifically, gB protein or UL32-homologue protein), for mycoplasma, adhesin protein, HMW-related protein, 40K protein 66K protein, 67K protein, and the like (sequences are described in International Patent Publication WO 94/23019).
Thus, it is preferred to integrate genes encoding these proteins. Such foreign genes are called heterologous antigen genes.
In order to express a heterologous antigen gene in HVT or MDV, it is necessary to integrate a promoter sequence into a region upstream of the heterologous antigen gene. The promoter used may be either a synthetic or natural promoter, and is not limited as long as it can effectively function as such in a transcription system owned by the cell infected with HVT or MDV.
As such a promoters, not only a promoter inherently owned by HVT or MDV but also a promoter or DNA derived from a virus other than HVT or MDV, a promoter derived from an eukaryotic or prokaryotic organism, or a synthetic promoter can be used in the present invention, as long as they satisfy the above requirements.
Specifically, there can be mentioned the thymidine kinase promoter of herpesvirus (Ross L. J., Gen. Virol. 74:371-377 (1993)), gB protein promoter (supra) of HVT or MDV, the IE promoter of human cytomegalovirus (HCMV) (Alting-Mess M. A., Nucleic Acids Res., 17:9494 (1989)), SV40 promoter (Gunning P., Proc. Natl. Acad. Sci., 84:4931-4835 (1987)), xcex2 actin promoter (supra, and Kost A. T., Nucleic Acids Res., 11:8287-8301 (1983)), xcex2-globin promoter (Spitzner J. R., Nucleic Acids Res., 18:1-11 (1990)), the LTR promoter of Rous sarcoma virus (Fiek A. et al., Nucleic Acids Res., 20:1785 (1992)), and the like. In addition, promoters of the structural proteins or the essential genes of HVT or MDV can also be used.
These may be integrated into the above-mentioned untranslated region in a manner such that a foreign antigen gene can be expressed under a control of a specific promoter, so as to construct a plasmid. The plasmid thus constructed may undergo recombination with a specific region of the HVT or MDV genome, thus resulting in the creation of a recombinant virus.
Construction of Recombinant Virus
The insertion of a foreign gene such as described above into an untranslated region of the genome of an avian infectious herpesvirus may be carried out according to a standard method. By way of example, HVT will be explained.
A plasmid in which a foreign antigen gene has been inserted into the HVT untranslated region obtained as above is introduced into an HVT-infected cell using electroporation, calcium phosphate, a lipofectin-based method, a gene gun method or the like. Because of the high efficiency of introduction, electroporation or a method employing lipofectin is preferred. When the amount of the plasmid to be introduced is in the range of 0.1 to 1000 xcexcg, the efficiency of generation of recombinant viruses by recombination between the homologous regions of HVT-DNA and the plasmid becomes high in cells into which the kiasmid has been introduced.
In order to select only a recombinant HVT thus created, one or a plurality of foreign genes are integrated into the untranslated region of the plasmid, and at least one of the genes used is an enzyme gene for color generation from a specific substrate. Examples of such an enzyme gene include xcex2-galactosidase gene.
A recombinant HVT containing the xcex2-galactosidase gene can be clearly distinguished from non-recombinant viruses because it develops a specific color by the addition of a substrate such as Bluogal. Therefore, by culturing cells infected with a virus that has integrated such a gene, in a culture medium supplemented with a specific substrate, it is possible to select virus-infected cells that develops dolor. By repeating this procedure, recombinant viruses can be purified.
Live Vaccines
Methods of preparing a live vaccine of the present invention include, but are not limited to, the following methods.
Cells infected with a recombinant virus of the present invention is infected to the cells (hereinafter referred to as the host cell) in which said virus can propagate, and grown. Then cells are detached from the wells using a scraper or with trypsin and separate the infected cells are separated from the supernatant by centrifugation.
As host cells, cells derived from chicken are preferred, and CEF (chick embryo fibroblast), chicken kidney cells, and the like, can be preferably used.
The infected cells thus obtained are suspended in a culture medium containing 10% dimethyl sulfoxide (DMSO) and stored frozen under liquid nitrogen. When they are used as a vaccine, the frozen product is dissolved in 100 volumes of phosphate buffer prior to use.
Stabilizers and other components for use in storing said infected cells under liquid nitrogen are not limited as long as they enable the virus-infected cells to grow stably and they are pharmaceutically acceptable.
Methods of inoculating a live vaccine of the present invention to poultry include, but are not limited to, a conventionally used subcutaneous injection, and is the same as used in the current HVT vaccines. The amount to be inoculated may also the same as the conventional vaccines.
The vaccine of the present invention can be used not only as a vaccine for infections of herpesviruses but also as a vaccine for diseases caused by an antigen gene inserted into the untranslated region by the pathogen from which the genes are derived. The vaccine of the present invention can be used as a useful recombinant HVT multi-valent vaccine.